Adjunctive use of the non-ionic surfactant Poloxamer 188 improves fetal dopaminergic cell survival and reinnervation in a neural transplantation strategy for Parkinson's disease.

Although neural transplantation of fetal dopaminergic cells is a promising therapy for Parkinson's disease, poor transplanted cell survival limits its efficacy. In the present study it was hypothesized that the use of Poloxamer 188 (P188), a non-ionic surfactant, during cell preparation and transplantation may protect cells from associated mechanical injury and thus improve transplanted cell survival in a rat model of Parkinson's disease. Fetal rat dopaminergic tissue was dissociated in media with or without P188 and then cultured for 1 week or transplanted into the striatum of rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal dopaminergic pathway. Fetal dopaminergic cell survival and reinnervation of the host brain were examined using tyrosine hydroxylase immunohistochemistry and stereological quantification. The number of surviving tyrosine hydroxylase-immunoreactive cells in vitro and in vivo was significantly increased by 2.2-fold by incubating fetal dopaminergic cells with P188 during tissue dissociation. Furthermore, the striatal reinnervation in parkinsonian rats that received intrastriatal transplants of P188-exposed dopaminergic cells was significantly enhanced (1.8-fold increase) compared with rats that received non-P188-treated cells. In conclusion, P188 protects fetal dopaminergic cells from mechanical injury by increasing cell survival and enhances dopaminergic fibre outgrowth into the transplanted striatum. Use of P188 may thus be an important adjunct to improve the clinical efficacy of neural transplantation for Parkinson's disease.

Differential effects of glial cell line-derived neurotrophic factor on A9 and A10 dopamine neuron survival in vitro.

Glial cell-line derived neurotrophic factor (GDNF) enhances dopamine (DA) cell survival and fiber outgrowth, and may be beneficial in enhancing cell restorative strategies for Parkinson's disease (PD). However, GDNF may have different roles for transplanted DA cell sub-types. The present in vitro study investigated the effect of GDNF on the survival of rat DA cells displaying a phenotype consistent with either the substantia nigra [A9 cells immunopositive for tyrosine hydroxylase (TH) and G-protein-gated inwardly rectifying potassium channel subunit 2 (GIRK2)] or with the ventral tegmental area [A10 cells immunopositive for TH and calbindin]. It was found that a single exposure of GDNF enhanced the number of DA cells of an A9 phenotype, without affecting DA cells of an A10 phenotype. Conversely, repeated GDNF exposure did not alter the survival of A9 phenotypic cells, but doubled the percentage of A10 cells. It was concluded that GDNF administration may affect dopaminergic cells differently depending on time and degree of GDNF exposure. For cell transplantation in PD, long-term GDNF administration may result in detrimental effects for transplanted A9 TH+ cells as this may introduce competition with A10 TH+ cells for survival and fiber outgrowth into the host striatum. These results may have important implications for clinical neural transplantation in PD.

Intracerebral co-transplantation of liposomal tacrolimus improves xenograft survival and reduces graft rejection in the hemiparkinsonian rat.

Immunosuppression remains a key issue in neural transplantation. Systemic administration of cyclosporin-A is currently widely used but has many severe adverse side effects. Newer immunosuppressive agents, such as tacrolimus (TAC) and rapamycin (RAPA), have been investigated for their neuroprotective properties on dopaminergic neurons. These drugs have been formulated into liposomal preparations [liposomal tacrolimus (LTAC) and liposomal rapamycin (LRAPA)] which retain these neuroprotective properties. Due to the slower release of the drugs from the liposomes, we hypothesized that co-transplantation of either LTAC or LRAPA within a xenogeneic cell suspension would increase cell survival and decrease graft rejection in the hemiparkinsonian rat, and that a combination of the two drugs may have a synergistic effect. 6-hydroxydopamine-lesioned rats were divided to four groups which received intra-striatal transplants of the following: 1) a cell suspension containing 400,000 fetal mouse ventral mesencephalic cells; 2) the cell suspension containing 0.63 microM LRAPA; 3) the cell suspension containing a dose of 2.0 microM LTAC; 4) the cell suspension containing 2.0 microM LTAC and 0.63 microM LRAPA. Functional recovery was assessed by amphetamine-induced rotational behavior. Animals were killed at 4 days or 6 weeks post-transplantation, and immunohistochemistry was performed to look at the expression of tyrosine hydroxylase and major histocompatibility complex classes I and II. Only the group receiving LTAC had a decrease in rotational behavior. This observation correlated well with significantly more surviving tyrosine hydroxylase immunoreactive cells compared with the other groups and significantly lower levels of immunorejection as assessed by major histocompatibility complex class I and II staining. This study has shown the feasibility of using local immunosuppression in xenotransplantation. These findings may be useful in optimizing immunosuppression in experimental neural transplantation in the laboratory and its translation into the clinical setting.

Erythropoietin and GDNF enhance ventral mesencephalic fiber outgrowth and capillary proliferation following neural transplantation in a rodent model of Parkinson's disease.

Low dopaminergic cell survival and suboptimal fiber reinnervation are likely major contributing factors for the limited benefits of neural transplantation in Parkinson's disease (PD) patients. Glial cell lined-derived neurotrophic factor (GDNF) has been shown to enhance dopaminergic cell survival and fiber outgrowth of the graft site as well as promote behavioral recovery in rodent models of PD, while erythropoietin (EPO) can produce dopaminergic neuroprotective effects against 6-hydroxydopamine (6-OHDA) exposure on cultured neurons and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice. The aim of this study was to determine if fetal ventral mesencephalic (FVM) tissue exposed to hibernation media containing a combination of GDNF and EPO could enhance dopaminergic graft survival, striatal reinnervation and functional recovery in a 6-OHDA rodent model of PD. FVM tissue was dissected from 14-day-old rat fetuses and placed for 6 days in hibernation media alone, and in hibernation media that received either a daily administration of GDNF, EPO or a combination of GDNF and EPO. Following hibernation, FVM cells were transplanted as a single cell suspension into the striatum of unilateral 6-OHDA-lesioned rats. Rotational behavioral assessment revealed animals that received FVM tissue exposed to GDNF, EPO or the combination of both drugs had accelerated functional recovery. Immunohistochemical and stereological assessment revealed a significant increase in graft fiber density and angiogenesis into the graft when compared with control. These findings suggest that the hibernation of FVM tissue in a combination of GDNF and EPO can enhance graft efficacy and may have important implications for tissue preparation protocols for clinical neural transplantation in PD.

Transplantation of bioreactor-produced neural stem cells into the rodent brain.

The development of new cell replacement strategies using neural stem cells (NSC) may provide an alternative and unlimited cell source for clinical neural transplantation in neurodegenerative diseases such as Parkinson's and Huntington's disease. The clinical application of neural transplantation using NSC will therefore depend upon the availability of clinical grade NSC that are generated in unlimited quantities in a standardized manner. In order to investigate the utility of NSC in clinical neural transplantation, undifferentiated murine NSC were first expanded for an extended period of time in suspension bioreactors containing a serum-free medium. Following expansion in suspension bioreactors, NSC were still able to differentiate in vitro into both astrocytes and neurons after exposure to brain-derived neurotrophic factor (BDNF), suggesting that bioreactor expansion does not alter cell lineage potentiality. Undifferentiated bioreactor-expanded NSC were then transplanted into the rodent striatum. Immunohistochemical examination revealed undifferentiated bioreactor-expanded NSC survived transplantation for up to 8 weeks and expressed the astrocytic immunohistochemical marker glial fibrillary acidic protein (GFAP), suggesting that the host striatal environment influences NSC cell fate upon transplantation. Moreover, no tumor formation was observed within the graft site, indicating that NSC expanded in suspension bioreactors for an extended period of time are a safe source of tissue for transplantation. Future studies should focus on predifferentiating NSC towards specific neuronal phenotypes prior to transplantation in order to restore behavioral function in rodent models of neurodegenerative disease.

A sequential intrastriatal dopaminergic graft strategy in the rodent model for Parkinson's disease: implications for graft survival and targeting.

Optimal placement of intrastriatal dopaminergic grafts is likely crucial to optimize clinical recovery in Parkinson's disease (PD). The target sites of dopaminergic grafts vary among clinical trials and may partially explain the variable results in clinical efficacy reported thus far. In this study we hypothesized that a subsequent dopaminergic graft may promote functional recovery following a suboptimal initial graft. To test this hypothesis, rats with unilateral 6-hydroxydopamine lesions of the right nigrostriatal pathway were randomly divided into three groups. The first group received 900,000 fetal nigral cells in the medial striatum only (n = 6). The second group received 900,000 cells in both the medial and lateral striatum simultaneously (1.8 million total; n = 8). The final group received a second graft of 900,000 cells in the lateral striatum 6 weeks following initial transplantation of a medial graft (n = 6). Amphetamine-induced circling behavior was significantly reduced in both simultaneous and sequential graft groups at 9 and 12 weeks following transplantation of the initial graft. However, no recovery was noted in the single medial graft group at those time points. Furthermore, increased survival of dopaminergic cells was observed in the lateral graft of sequentially grafted animals compared with the medial graft. We conclude that a well-positioned subsequent graft can restore function in animals with a suboptimal initial graft and that the initial graft may improve survival of the second graft. These results are further discussed in relation to their important clinical implication for neural transplantation in PD.

Liposomal tacrolimus administered systemically and within the donor cell suspension improves xenograft survival in hemiparkinsonian rats.

The most widely used immunosuppressant in neural transplantation is cyclosporine- A (CsA). However, CsA has significant toxic effects when administered systemically. Tacrolimus (FK506), is a more potent immunosuppressant than CsA and can be prepared in lipid micelles (LTAC). This liposomal preparation allows for the administration of tacrolimus to the site of transplantation, possibly reducing the systemic side effects of immunosuppression. In this study we investigated the ability of LTAC to promote graft survival in hemiparkinsonian rats implanted with fetal mouse xenografts when LTAC is administered systemically to the host, when added to the donor cell suspension, or in combination. Rats with unilateral 6-hydroxydopamine lesions were transplanted with 800,000 fetal mouse ventral mesencephalic (VM) cells and were randomly divided into four groups. Group 1 was not immunosuppressed; Group 2 received daily systemic injections of LTAC; Group 3 received LTAC within the cell suspensions of mouse VM cells; and Group 4 received LTAC in the cell suspensions along with daily systemic administration of LTAC. Transplanted rats were assessed for rotational behavior 3 and 6 weeks posttransplantation. Cell survival was assessed using tyrosine hydroxylase (TH) immunohistochemistry. A significant reduction in rotational scores was observed only in the group of animals receiving the combination of LTAC-treated donor cells and systemic LTAC administration. This functional improvement correlated with a significantly greater survival of TH-immunoreactive cells in this group of animals. The other groups had poor cell survival and no significant functional improvement. We conclude that a combination of systemic immunosuppression and treatment of the cell suspension with LTAC may be a superior strategy to optimize xenograft survival. This strategy may have important implications for clinical neural transplantation.

Enhancement of sensorimotor behavioral recovery in hemiparkinsonian rats with intrastriatal, intranigral, and intrasubthalamic nucleus dopaminergic transplants.

One of the critical variables that influences the efficacy of clinical neural transplantation for Parkinson's disease (PD) is optimal graft placement. The current transplantation paradigm that focuses on ectopic placement of fetal grafts in the striatum (ST) fails to reconstruct the basal ganglia circuitry or normalize neuronal activity in important basal ganglia structures, such as the substantia nigra (SN) and the subthalamic nucleus (STN). The aim of this study was to investigate a multitarget neural transplantation strategy for PD by assessing whether simultaneous dopaminergic transplants in the ST, SN, and STN induce functional recovery in hemiparkinsonian rats. Forty-six female Wistar rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway were randomly divided into eight groups and received lesions only or injections of 900,000 embryonic rat ventral mesencephalic cells in the (1) ST, (2) SN, (3) STN, (4) ST and SN, (5) ST, SN, and STN, (6) ST and STN, or (7) SN and STN. The number of cells transplanted was equally divided among grafting sites. Animals with two grafts received 450,000 cells in each structure, and animals with three grafts received 300,000 cells per structure. Recovery was assessed by amphetamine-induced rotations and the stepping tests. Graft survival was assessed using tyrosine hydroxylase immunohistochemistry. At 8 weeks after transplantation, simultaneous dopaminergic transplants in the ST, SN, and STN induced significant improvement in rotational behavior and stepping test scores. Intrastriatal transplants were associated with significant recovery of rotational asymmetry, whereas SN and STN transplants were associated with improved forelimb function scores. These results suggest that restoration of dopaminergic activity to multiple basal ganglia targets, such as the ST and SN, or the ST and STN, promotes a more complete functional recovery of complex sensorimotor behaviors. A multitarget transplant strategy aimed at optimizing dopaminergic reinnervation of the basal ganglia may be crucial in improving clinical outcomes in PD patients.

Neutralizing antibodies against neurite growth inhibitor NI-35/250 do not promote regeneration of sensory axons in the adult rat spinal cord.

Neutralization of the myelin-associated neurite growth inhibitors NI-35 and NI-250 by IN-1 antibodies can promote axonal regeneration of several types of central nervous neurons. Here, we investigated in adult rats whether IN-1 can promote regeneration of ascending sensory axons across a peripheral nerve bridge back into the spinal cord. IN-1 was administered by hybridoma cells injected in the cerebral cortex or thoracic cord, its presence confirmed in tissue sections and cerebrospinal fluid, and its effectiveness demonstrated in co-cultures of oligodendrocytes and sensory neurons. With a two week infusion of control vehicle into the dorsal spinal cord 3 mm rostral to the nerve graft, only 3+/-2% of the anterogradely labeled sensory fibers present at the rostral end of the nerve graft had grown up to 0.5 mm, but not farther into the spinal cord. A similar limited extent of regeneration was seen with IN-1 or with infusion of Dantrolene, an inhibitor of NI-35/250 activity in vitro. With infusion of nerve growth factor rostral to the nerve graft, 40% of the fibers at the rostral end of the graft were found at 0.5 mm, 34% at 1 mm, 24% at 2 mm and 14% at 3 mm (the infusion site) into the spinal cord. Treatment with IN-l antibodies did not enhance the growth-promoting effects of nerve growth factor. We suggest that the neurite growth inhibitors NI-35 or NI-250 do not play a major inhibitory role in the regeneration of the ascending sensory fibers across a nerve bridge and back into the spinal cord of the adult rat.

Simultaneous intrastriatal and intranigral dopaminergic grafts in the parkinsonian rat model: role of the intranigral graft.

The current transplantation strategy in experimental and clinical Parkinson's disease (PD) has been to place nigral dopaminergic grafts not in their ontogenic site (substantia nigra) but in their target area (striatum). Although intrastriatal dopaminergic grafts are capable of reinnervating the striatum, they fail to reinnervate the nigra, which may be an important factor limiting the efficacy of fetal tissue transplantation in parkinsonian patients. We have previously shown that simultaneous intrastriatal and intranigral dopaminergic grafts (double grafts) may provide a more complete restoration of the nigrostriatal circuitry (Mendez et al. [1996] J Neurosci 16:7216-7227; Mendez and Hong [1997] Brain Res 778:194-205). In the present study, we investigated the contribution of the intranigral graft to functional recovery in double-grafted hemiparkinsonian rats. Twenty Wistar rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway were divided into two groups and received either double grafts (n = 10) or intrastriatal grafts alone (n = 10). Following transplantation, both intrastriatally and double-grafted animals had a significant decrease in rotational behavior. However, only animals with double grafts exhibited a significant increase in contralateral adjusting step performance. The intranigral graft was subsequently lesioned by a second 6-OHDA injection. Following the second lesion, animals with double grafts exhibited a significant reversal of rotational behavior and a 51% reduction in contralateral adjusting step performance. The reversal in functional recovery correlated with a significant loss of intranigral grafted neurons. These results suggest that the intranigral graft has an important role in the functional recovery of double-grafted animals. Restoration of dopaminergic innervation to both the nigra and the striatum may be crucial for optimizing graft efficacy and may be a superior strategy in neural transplantation for PD.

Intrastriatal and intranigral grafting of hNT neurons in the 6-OHDA rat model of Parkinson's disease.

The clinical findings on neural transplantation for Parkinson's disease (PD) reported thus far are promising but many issues must be addressed before neural transplantation can be considered a routine therapeutic option for PD. The future of neural transplantation for the treatment of neurological disorders may rest in the discovery of a suitable alternative cell type for fetal tissue. One such alternative may be neurons derived from a human teratocarcinoma (hNT). hNT neurons have been shown to survive and integrate within the host brain following transplantation and provide functional recovery in animal models of stroke and Huntington's disease. In this study, we describe the transplantation of hNT neurons in the substantia nigra (SN) and striatum of the rat model for PD. Twenty-seven rats were grafted with one of three hNT neuronal products; hNT neurons, hNT-DA neurons, or lithium chloride (LiCl) pretreated hNT-DA neurons. Robust hNT grafts could be seen with anti-neural cell adhesion molecule and anti-neuron-specific enolase immunostaining. Immunostaining for tyrosine hydroxylase (TH) expression revealed no TH-immunoreactive (THir) neurons in any animals with hNT neuronal grafts. THir cells were observed in 43% of animals with hNT-DA neuronal grafts and all animals with LiCl pretreated hNT-DA neuronal grafts (100%). The number of THir neurons in these animals was low and not sufficient to produce significant functional recovery. In summary, this study has demonstrated that hNT neurons survive transplantation and express TH in the striatum and SN. Although hNT neurons are promising as an alternative to fetal tissue and may have potential clinical applications in the future, further improvements in enhancing TH expression are needed.

Reconstruction of the nigrostriatal pathway by simultaneous intrastriatal and intranigral dopaminergic transplants.

The main strategy in experimental and clinical neural transplantation in Parkinson's disease has been to place fetal nigral grafts not in their ontogenic site (substantia nigra) but in their target area (striatum). The reason for this ectopic placement is the apparent inability of nigral grafts placed in the ventral mesencephalon (VM) of the adult host to grow axons for long distances that are capable of reaching the ipsilateral striatum and thus restoring the nigrostriatal pathway. The present study demonstrates for the first time that simultaneous dopaminergic transplants (double grafts) placed in the substantia nigra and ipsilateral striatum of rats bearing unilateral 6-hydroxydopamine lesions reconstruct the dopaminergic nigrostriatal pathway in the adult rat brain. Numerous tyrosine hydroxylase-immunoreactive axons were observed arising from the intranigral graft and growing rostrally along the internal capsule and medial forebrain bundle to reinnervate the ipsilateral striatum, which also had received a dopaminergic graft. These double grafts achieved not only greater striatal reinnervation than the standard intrastriatal graft but also a faster and more complete rotational recovery to amphetamine challenge 6 weeks after transplantation. These results suggest strongly that embryonic nigral transplants implanted in the striatum are capable of promoting growth and providing guidance to axons arising from a dopaminergic graft placed homotopically in the VM, resulting in restoration of the dopaminergic nigrostriatal projection. Reconstruction of the nigrostriatal pathway by double grafts may not only achieve substantial striatal reinnervation but may also contribute to the reestablishment of dopaminergic regulation of the nigrostriatal circuitry.

A substrate amplification system for enzyme-linked immunoassays. II. Demonstration of its applicability for measuring anti-DNA antibodies.

We have recently described a substrate amplification system, based on the method of Self, which increases the sensitivity of alkaline phosphatase (AP)-dependent enzyme-linked immunosorbent assays (ELISA) by a factor of 30-50. This increase is achieved by having AP, the primary enzyme, produce an activator for a secondary enzyme-substrate system, within which marked amplification occurs. We have now demonstrated that this amplification method can be applied to the measurement of human antibodies to DNA. The sensitivity is greater by a factor of 10 than the conventional method, which uses p-nitrophenyl phosphate (p-NPP) as substrate. On replicate assays the method is reproducible, with a coefficient of variation of less than 0.1. This great increase in sensitivity should be of value in conserving specimens of serum and in screening monoclonal antibodies.

Abnormal responses to ingested substances in murine systemic lupus erythematosus: apparent effect of a casein-free diet on the development of systemic lupus erythematosus in NZB/W mice.

To assess the development of oral tolerance to casein in NZB/W female mice, they must be bred and raised on a casein free diet. We examined the specific immune responses of the mice to the long term experimental feeding of casein. Twelve of fifteen casein free mice were still alive at 10 months of age, although by this age only 1/10 mice eating the normal diet was still alive. The casein free mice had markedly less anti-DNA antibody, their IgM to IgG antinative DNA switch was delayed and deposits of immunoreactants in the glomeruli were greatly decreased. The reason for this apparent effect of the removal of casein from the diet is unknown; however, immunostimulatory and endorphin-like regions have recently been reported in casein.

A substrate amplification system for enzyme-linked immunoassays. Demonstration of its general applicability to ELISA systems for detecting antibodies and immune complexes.

Self recently described a substrate system for alkaline phosphatase (AP)-dependent ELISAs which markedly increased sensitivity, compared to using p-nitrophenyl phosphate. This increase is achieved by having AP, the primary enzyme, produce an activator for a secondary enzyme-substrate system, within which marked amplification occurs. We adapted this technique to study antibodies to casein, bovine serum albumin, ovalbumin, and cardiolipin in the sera of patients with systemic lupus erythematosus (SLE) and normal individuals. The new substrate system yielded titres 30-50-fold higher than those with p-nitrophenyl phosphate (Sigma 104, p-NPP). In addition, when used in a solid-phase C1q binding assay we were able to use a 1 : 100,000 dilution of AP-conjugated anti-human IgG with the amplified substrate, compared to the 1 : 1000 dilution needed with p-NPP. This system is extremely valuable because of its flexibility. It can either be very sparing of limited samples, or if the added sensitivity is not needed, 100-fold less AP conjugate may be used. Thus rare or expensive conjugates can be significantly conserved.

Failure of oral tolerance in (NZB X NZW)F1 mice is antigen specific and appears to parallel antibody patterns in human systemic lupus erythematosus (SLE).

Primary oral antigen exposure normally induces mucosal immunity and an active suppression of the systemic immune response. Patients with systemic lupus erythematosus (SLE) have increased antibodies to bovine gamma-globulin (BGG), which suggested a possible failure of oral tolerance in SLE. We examined this possibility in murine lupus. NZB/W females were fed BGG or saline and were subsequently immunized ip. Primary and secondary responses were assessed. At 1 month of age the mice tolerized normally in response to feeding with BGG but, at 4 months of age, not only did they not tolerize, the mice fed BGG had a 5- to 7-fold higher response to parenteral immunization than did the saline-fed mice. Control strain mice tolerized normally at both ages (a 5- to 10-fold lower response). Conversely, when fed ovalbumin, NZB/W females tolerized normally at both 1 and 4 months of age, and patients with SLE had normal levels of antibody to this antigen. However, we also found increased levels of antibodies to bovine casein in SLE patients, and found that NZB/W mice failed to orally tolerize with this antigen at either 1 or 4 months of age. Thus, the failure of oral tolerance in the NZB/W mice appears to be antigen specific and age dependent and, at least with respect to these three antigens, appears to parallel the antibody patterns seen in human SLE.

Orally induced tolerance to casein in mice on normal mouse chow.

We examined the ability of bovine casein to induce oral tolerance in BDF1 mice raised on a normal mouse chow diet. Giving 20 mg casein 1 X or 4 X by intragastric tube or for 28 days at 1 mg/ml in drinking water failed to have a significant effect (P greater than 0.2) on the subsequent immune response to parenteral casein, suggesting that casein was not an effective oral tolerogen. However, we discovered that most normal mouse chow contains casein. When BDF1 mice were raised on a casein-free diet, and treated as above, they showed marked suppression of subsequent responses to parenteral casein (P less than 0.01). Our results indicated that mice on the normal diet were presuppressed by the dietary exposure. Examination of several other normal mouse chows revealed that they all contained casein as a protein source. A possible effect of prior exposure must be considered in all experimental animal studies involving immune responses to antigens which may be present in the environment.